What is the effect of the side chains colored green on the TEA binding?

The side chains colored green, when mutated to cysteine, are modified by cysteine-reactive agents whether or not the channel gate is open, whereas those colored pink react only when the channel is open (16).

What is the general property of all selective ion channels?

On the basis of functional measurements, the same concept of destabilization by multiple ion occupancy has been proposed for Ca21 channels (22) and for K1 channels (23) and may be a general property of all selective ion channels.

What was the only molecule in the structure that was not associated with an ion peak?

A single water molecule (the only one modeled in the structure) located between the two K1 ions in the selectivity filter was justified by the presence of a strong electron density peak in the experimental map, which was never associated with an ion peak in the difference Fourier maps (19).

What is the effect of the side chain at the base of the selectivity filter?

The mustard-colored side chain at the base of the selectivity filter affects TEA binding from the intracellular solution [the internal TEA site (15)].

How did the authors determine the ion position of the K1 channel?

The authors also used K1 ion positions defined by difference Fourier analysis (Figs. 6 and 8A, yellow density) and their knowledge of alkali metal cation coordination in small molecules.

What is the structure of the selectivity filter?

The ions are located at opposite ends of the selectivity filter, separated by about 7.5 Å, roughly the average distance between K1 ions in a 4 M KCl solution, and in the selectivity filter there are no intervening Cl2 anions to balance the charge.

What is the effect of the second ion on the selectivity filter?

when a second ion enters, the attractive force between a K1 ion and the selectivity filter becomes perfectly balanced by the repulsive force between ions, and this is what allows conduction to occur.

What is the role of the cerebellum in the acquisition and retention of delay classical conditioning?

Studies in the rabbit have shown that the cerebellum is essential for both the acquisition and retention of delay classical conditioning (4) and that no other forebrain structure, including the hippocampus, is required (5).

Why is the selectivity filter held open?

The structure reveals that the selectivity filter is held open as if to prevent it from accommodating a Na1ion with its smaller radius.

(Open Access) The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity (1998) | Declan A. Doyle

Q: What contributions have the authors mentioned in the paper "The structure of the potassium channel: molecular basis of k conduction and selectivity" ?

In this paper, the authors used X-ray analysis with data to 3.2 angstroms reveals that four identical subunits create an inverted teepee, or cone, cradling the selectivity filter of the pore in its outer end.

Q: What is the amino acid sequence of the KcsA channel?

Although the KcsA K1 channel is a two membrane-spanning K1 channel, its amino acid sequence is actually closer to those of eukaryotic six membranespanning K1 channels.

The Structure of the Potassium

Channel: Molecular Basis of K

Conduction and Selectivity

Declan A. Doyle, Joa˜ o Morais Cabral, Richard A. Pfuetzner,

Anling Kuo, Jacqueline M. Gulbis, Steven L. Cohen,

Brian T. Chait, Roderick MacKinnon*

The potassium channel from Streptomyces lividans is an integral membrane protein with

sequence similarity to all known K

channels, particularly in the pore region. X-ray

analysis with data to 3.2 angstroms reveals that four identical subunits create an inverted

teepee, or cone, cradling the selectivity filter of the pore in its outer end. The narrow

selectivity filter is only 12 angstroms long, whereas the remainder of the pore is wider

and lined with hydrophobic amino acids. A large water-filled cavity and helix dipoles are

positioned so as to overcome electrostatic destabilization of an ion in the pore at the

center of the bilayer. Main chain carbonyl oxygen atoms from the K

channel signature

sequence line the selectivity filter, which is held open by structural constraints to co-

ordinate K

ions but not smallerNa

ions. The selectivity filter contains two K

ions about

7.5 angstroms apart. This configuration promotes ion conduction by exploiting electro-

static repulsive forces to overcome attractive forces between K

ions and the selectivity

filter. The architecture of the pore establishes the physical principles underlying selective

conduction.

Potassium ions diffuse rapidly across cell

membranes through proteins called K

channels. This movement underlies many

fundamental biological processes, includ-

ing electrical signaling in the nervous sys-

tem. Potassium channels use diverse

mechanisms of gating (the processes by

which the pore opens and closes), but they

all exhibit very similar ion permeability

characteristics (1). All K

channels show

a selectivity sequence of K

' Rb

, whereas permeability for the smallest

alkali metal ions Na

and Li

is immea-

surably low. Potassium is at least 10,000

times more permeant than Na

, a feature

that is essential to the function of K

channels. Potassium channels also share a

constellation of permeability characteris-

tics that is indicative of a multi-ion

conduction mechanism: The flux of ions

in one direction shows high-order cou-

pling to flux in the opposite direction, and

ionic mixtures result in anomalous con-

duction behavior (2). Because of these

properties, K

channels are classified

as “long pore channels,” invoking the

notion that multiple ions queue inside a

long, narrow pore in single file. In

addition, the pores of all K

channels

can be blocked by tetraethylammonium

(TEA) ions (3).

Molecular cloning and mutagenesis ex-

periments have reinforced the conclusion

that all K

channels have essentially the

same pore constitution. Without exception,

all contain a critical amino acid sequence,

the K

channel signature sequence. Muta-

tion of these amino acids disrupts the chan-

nel’s ability to discriminate between K

and Na

ions (4).

Two aspects of ion conduction by K

channels have tantalized biophysicists for

the past quarter century. First, what is the

chemical basis of the impressive fidelity

with which the channel distinguishes be-

tween K

and Na

ions, which are feature-

less spheres of Pauling radius 1.33 Å and

0.95 Å, respectively? Second, how can K

channels be so highly selective and at the

same time, apparently paradoxically, exhib-

it a throughput rate approaching the diffu-

sion limit? The 10

margin by which K

selected over Na

implies strong energetic

interactions between K

ions and the pore.

And yet strong energetic interactions seem

incongruent with throughput rates up to

ions per second. How can these two

essential features of the K

channel pore be

reconciled?

Potassium Channel Architecture

The amino acid sequence of the K

chan-

nel from Streptomyces lividans (KcsA K

channel) (5) is similar to that of other K

channels, including vertebrate and inverte-

brate voltage-dependent K

channels, ver-

tebrate inward rectifier and Ca

-activated

channels, K

channels from plants and

bacteria, and cyclic nucleotide-gated cation

channels (Fig. 1). On the basis of hydro-

phobicity analysis, there are two closely

related varieties of K

channels, those con-

taining two membrane-spanning segments

per subunit and those containing six. In all

cases, the functional K

channel protein is

a tetramer (6), typically of four identical

subunits (7). Subunits of the two mem-

brane-spanning variety appear to be short-

ened versions of their larger counterparts, as

if they simply lack the first four membrane-

spanning segments. Although the KcsA K

channel is a two membrane-spanning K

channel, its amino acid sequence is actually

closer to those of eukaryotic six membrane-

spanning K

channels. In particular, its

sequence in the pore region, located be-

tween the membrane-spanning stretches

and containing the K

channel signature

sequence, is nearly identical to that found

in the Drosophila (Shaker) and vertebrate

voltage-gated K

channels (Fig. 1). In an

accompanying paper, through a study of the

KcsA K

channel interaction with eukary-

otic K

channel toxins, we confirm that

the KcsA pore structure is indeed very sim-

ilar to that of eukaryotic K

channels and

that its structure is maintained when it is

removed from the membrane with deter-

gent (8).

We have determined the KcsA K

channel structure from residue position 23

to 119 by x-ray crystallography (Table 1).

The cytoplasmic carboxyl terminus (resi-

dues 126 to 158) was removed in the prep-

aration and the remaining residues were

disordered. The KcsA K

channel crystals

are radiation-sensitive and the diffraction

pattern is anisotropic, with reflections ob-

served along the best and worst directions

at 2.5 Å and 3.5 Å Bragg spacings, respec-

tively. By data selection, anisotropy correc-

tion, introduction of heavy atom sites by

site-directed mutagenesis, averaging, and

solvent flattening, an interpretable electron

density map was calculated (Fig. 2, A

through C). This map was without main

chain breaks and showed strong side chain

density (Fig. 2C). The model was refined

with data to 3.2 Å (the data set was 93 %

complete to 3.2 Å with 67% completeness

between 3.3 Å and 3.2 Å), maintaining

highly restrained stereochemistry and keep-

ing tight noncrystallographic symmetry re-

straints. The refinement procedure was

D. A. Doyle, R. A. Pfuetzner, A. Kuo, and R. MacKinnon

are in the Laboratory of Molecular Neurobiology and Bio-

physics and the Howard Hughes Medical Institute, Rock-

efeller University, 1230 York Avenue, New York, NY

10021, USA. J. M. Cabral and J. M. Gulbis are in the

Laboratory of Molecular Neurobiology and Biophysics,

Rockefeller University, 1230 York Avenue, New York, NY

10021, USA. S. L. Cohen and B. T. Chait are in the

Laboratory of Mass Spectrometry and Gaseous Ion

Chemistry, Rockefeller University, 1230 York Avenue,

New York, NY 10021, USA.

*To whom correspondence should be addressed. E-mail:

mackinn@rockvax.rockefeller.edu

RESEARCH ARTICLES

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SCIENCE

VOL. 280

3 APRIL 1998 69

monitored by minimizing the value R-free

(29.0%) and its separation from R-crystal-

lographic (28.0%). The presence of four

molecules (subunits) in the asymmetric unit

of the crystal provides a very significant

enhancement of the accuracy of the crys-

tallographic analysis; first, by enabling av-

eraging of the electron density over four

crystallographically independent regions of

the multiple isomorphous replacement

(MIR) map, and second, by providing a

powerful set of constraints on the atomic

model during refinement (9).

The K

channel is a tetramer with four-

fold symmetry about a central pore (Fig. 3,

A and B). Like several other membrane

proteins, it has two layers of aromatic amino

acids positioned to extend into the lipid

bilayer, presumably near the membrane-wa-

ter interfaces (Fig. 3C) (10). Each subunit

has two transmembrane a-helices connect-

ed by the roughly 30 amino acid pore re-

gion, which consists of the turret, pore he-

lix, and selectivity filter (Fig. 3, A and B).

A subunit is inserted into the tetramer such

that one transmembrane helix (inner helix)

faces the central pore while the other (outer

helix) faces the lipid membrane. The inner

helices are tilted with respect to the mem-

brane normal by about 25° and are slightly

kinked, so that the subunits open like the

petals of a flower facing the outside of the

cell. The open petals house the structure

formed by the pore region near the extra-

cellular surface of the membrane. This re-

gion contains the K

channel signature

sequence, which forms the selectivity filter

(4). The essential features of subunit pack-

ing can be appreciated by viewing the rela-

tion between the four inner helices and the

four pore helices (Fig. 3D). The four inner

helices pack against each other as a bundle

near the intracellular aspect of the mem-

brane, giving the appearance of an inverted

teepee. The pore helices are slotted in be-

tween the poles of the teepee and are di-

rected, with an amino-to-carboxyl sense,

toward a point near the center of the chan-

nel (Fig. 3, A, B, and D). This pore helix

arrangement provides many of the intersub-

unit contacts that hold the tetramer togeth-

er and, as discussed below, is also critical in

the operation of the ion conduction pore.

Sequence conservation among K

chan-

nels (including ones with two and six mem-

brane-spanning segments), as well as cyclic

nucleotide-gated cation channels, is stron-

gest for the amino acids corresponding to

the pore region and the inner helix. Even

and Ca

channels show distant relat-

edness over these segments. The teepee ar-

chitecture of the K

channel pore likely

will be a general feature of all of these

cation channels, with four inner helices

arranged like the poles of a teepee, four pore

helices, and a selectivity filter—tuned to

select the appropriate cation—located close

to the extracellular surface.

This structure of the KcsA K

channel

is in excellent agreement with results from

functional and mutagenesis studies on Shak-

er and other eukaryotic K

channels (Fig.

4). The pore-region of K

channels was

first defined with pore-blocking scorpion

toxins (11). These inhibitors interact with

amino acids (Fig. 4, white) comprising the

broad extracellular-facing entryway to the

pore (12). The impermeant organic cation

TEA blocks K

channels from both sides of

the membrane at distinct sites (13). Amino

acids interacting with externally and inter-

nally applied TEA are located just external

to (Fig. 4, yellow) and internal to (Fig. 4,

mustard) the structure formed by the signa-

ture sequence amino acids (14, 15). Alter-

ation of the signature sequence amino acids

(Fig. 4, red main chain atoms) disrupts K

selectivity (4). Amino acids close to the

intracellular opening on the Shaker K

channel map to the inner helix on the

KcsA K

channel (16). Interestingly, expo-

sure to the cytoplasm of the region above

the inner helix bundle (Fig. 4, pink side

chains) requires an open voltage-dependent

gate, whereas the region at or below the

bundle (Fig. 4, green side chains) is exposed

whether or not the gate was open. The

correlation between the transition zone for

gate-dependent exposure to the cytoplasm

in the Shaker K

channel and the inner

helix bundle in this structure has im-

plications for mechanisms of gating in K

channels.

General Properties of the Ion

Conduction Pore

As might have been anticipated for a cation

channel, both the intracellular and extra-

cellular entryways are negatively charged by

acidic amino acids (Fig. 5A, red), an effect

that would raise the local concentration of

cations while lowering the concentration of

anions. The overall length of the pore is 45

Å, and its diameter varies along its distance

(Fig. 5B). From inside the cell (bottom) the

pore begins as a tunnel 18 Å in length (the

internal pore) and then opens into a wide

cavity (;10 Å across) near the middle of

the membrane. A K

ion could move

throughout the internal pore and cavity and

still remain mostly hydrated. In contrast,

the selectivity filter separating the cavity

kcsa TYPRALWWSVETATTVGYGDLY..PVTLWGRLVAVVVMVAGITSFGLVTAALATWFVGRE

kch SLMTAFYFSIETMSTVGYGDIV..PVSESARLFTISVIISGITVFATSMTSIFGPLIRGG

clost SLGNALWWSFVTITTVGYGDIS..PSTPFGRVIASILMLIGIGFLSMLTGTISTFFISKK

Shaker SIPDAFWWAVVTMTTVGYGDMT..PVGFWGKIVGSLCVIAGVLTIALPVPVIVSNFNYFY

hKv1.1 SIPDAFWWAVVSMTTVGYGDMY..PVTIGGKIVGSLCAIAGVLTIALPVPVIVSNFNYFY

hDRK SIPASFWWATITMTTVGYGDIY..PKTLLGKIVGGLCCIAGVLVIALPIPIIVNNFSEFY

Parame QYLHSLYWSIITMTTIGYGDIT..PQNLRERVFAVGMALSAVGVFGYSIGNINSIYAEWS

Celegans SIPLGLWWAICTMTTVGYGDMT..PHTSFGRLVGSLCAVMGVLTIALPVPVIVSNFAMFY

mSlo TYWECVYLLMVTMSTVGYGDVY..AKTTLGRLFMVFFILGGLAMFASYVPEIIELIGNRK

cal_act NFLGAMWLISITFLSIGYGDMV..PHTYCGKGVCLLTGIMGAGCTALVVAVVARKLELTK

AKT1 RYVTSMYWSITTLTTVGYGDLH..PVNTKEMIFDIFYMLFNLGLTAYLIGNMTNLVVHGT

herg KYVTALYFTFSSLTSVGFGNVS..PNTNSEKIFSICVMLIGSLMYASIFGNVSAIIQRLY

romk GMTSAFLFSLETQVTIGYGFRFVTEQCATAIFLLIFQSILGVIINSFMCGAILAKISRPK

hgirk GFVSAFLFSIETETTIGYGYRVITDKCPEGIILLLIQSVLGSIVNAFMVGCMFVKISQPK

olCNG EYIYCLYWSTLTLTTIG..ETPP.PVKDEEYLFVIFDFLIGVLIFATIVGNVGSMISNMN

rodCNG KYVYSLYWSTLTLTTIG..ETPP.PVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISNMN

OUTER HELIX

INNER HELIX

PORE

HELIX

PORE REGION

61 70 80 90 100 110

Fig. 1. Sequence alignment of selected K

channels and cyclic nucleotide-gated channels. The

numbering and secondary structural elements for the Streptomyces lividans K

channel (KcsA) is given

above the sequences. Selectivity filter, red; lining of the cavity and inner pore, blue; residues in which the

nature of the side chain is preserved (.50% similarity), grey. The sequences are: KcsA, Streptomyces

lividans, accession number (acc) PIR S60172; kch, Escherichia coli, acc GenBank U24203; clost,

Clostridium acetobutylicum (Genome Therapeutics Corp.); Shaker, Drosophila melanogaster, acc PIR

S00479; hKv1.1, Homo sapiens, acc Swissprot Q09470; hDRK, H. sapiens, acc PIR S31761;

Parame, Paramecium tetraaurelia, acc GenBank U19908; Celegans, Caenorhabditis elegans, acc

GenBank AF005246; mSlo, Mus musculus, acc PIR A48206; cal_act, H. sapiens, acc GenBank

AF031815; AKT1, Arabidopsis thaliana, acc PIR S62694; herg, H. sapiens, acc PIR I38465; romk,

Rattus norvegicus, acc Swissprot P35560; hgirk, H. sapiens, acc GenBank S78684; olCNG, H. sapi-

ens, acc Swissprot Q16280; rodCNG, H. sapiens, acc PIR A42161. The last two sequences, separate

from the rest, are from cyclic nucleotide–gated channels, which are not K

selective.

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VOL. 280

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www.sciencemag.org70

from the extracellular solution is so narrow

that a K

ion would have to shed its hy-

drating waters to enter. The chemical com-

position of the wall lining the internal pore

and cavity is predominantly hydrophobic

(Fig. 5A, yellow). The selectivity filter, on

the other hand, is lined exclusively by polar

main chain atoms belonging to the signa-

ture sequence amino acids. The distinct

mechanisms operating in the cavity and

internal pore versus the selectivity filter will

be discussed below, but first we introduce

the determination of K

ion positions in

the pore.

Potassium channels exclude the smaller

alkali metal cations Li

(radius 0.60 Å) and

(0.95 Å) but allow permeation of the

larger members of the series Rb

(1.48 Å)

and Cs

(1.69 Å). In fact, Rb

is nearly a

perfect K

(1.33 Å) analog because its size

and permeability characteristics are very

similar to those of K

. Because they are

more electron dense than K

,Rb

and Cs

allow visualization of the locations of per-

meant ions in the pore. By difference elec-

tron density maps calculated with data from

crystals transferred into Rb

-containing

(Fig. 6a) or Cs

-containing (Fig. 6b) solu-

tions, multiple ions are well defined in the

pore. The selectivity filter contains two

ions (inner and outer ions) located at op-

posite ends, about 7.5 Å apart (center to

center). In the Rb

difference map, there

actually are two partially separated peaks at

the inner aspect of the selectivity filter.

These peaks are too close to each other (2.6

Å) to represent two simultaneously occu-

pied ion binding sites. We suspect that they

represent a single ion (on average) in rapid

equilibrium between adjacent sites. The

single inner ion peak in the Cs

difference

map undoubtedly reflects the lower resolu-

tion at which the map was calculated (to 5

Å for Cs

versus 4.0 Å for Rb

), because

the Rb

difference map, when calculated at

the same lower resolution, also shows only a

single peak at the Cs

position. The Rb

positions correspond to strong peaks (pre-

Fig. 2. Experimental electron density map. Ste-

reoviews of the experimental electron-density

map contoured at 1 s covering nearly an entire

subunit (removed from the tetramer) of the final

model. The map was calculated at 3.2 Å resolu-

tion with the following Fourier coefficients: native-

sharpened amplitudes and MIR solvent flattened

averaged phases. (A) Foreground: map showing

inner helix, loop structures and selectivity filter;

background: the pore helix and outer helix. CPK

spheres show positions of mercury atoms used as

residue markers (from the top, marked residues

are Leu

, Leu

and Val

). (B) Alternative view.

Foreground: pore helix and part of outer helix;

background: selectivity filter and turret. CPK

sphere marks position of Ala

.(C) Close up view

of electron density.

RESEARCH ARTICLES

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SCIENCE

VOL. 280

3 APRIL 1998 71

sumably, K

ions) in a high contour native

electron density map (not shown). Thus,

the selectivity filter contains two K

ions.

A third weaker peak is located below the

selectivity filter at the center of the large

cavity in the Rb

difference map (Fig. 6a,

cavity ion) and in the Cs

difference map

at a lower contour (not shown). Electron

density at the cavity center is prominent in

MIR maps, even prior to averaging (Fig. 6c,

lower diffuse peak). The difference electron

density maps show this to be related to the

presence of one or more poorly localized

cations situated at least 4 Å away from

the closest protein groups.

The Cavity and Internal Pore

Why is there a 10 Å diameter cavity in the

center of the channel with an ion in it

(Fig. 5B and Fig. 6)? Electrostatic calcula-

tions show that when an ion is moved

along a narrow pore through a membrane,

it must cross an energy barrier that is

maximum at the membrane center (17).

The electrostatic field emanating from a

cation polarizes its environment, bringing

the negative ends of dipoles closer to it

and thereby stabilizing it. At the bilayer

center, the polarizability of the surround-

Table 1. Summary of data collection and refinement statistics. Crystals

(space group C2: a 5 128.8 Å, b 5 68.9 Å, c 5 112.0 Å, b5124.6°) were

flash-frozen by being transferred directly from the crystal mother liquor to a

stream of boiled-off nitrogen (24). Because crystals of the mutant L90C

diffracted significantly better than wild-type protein crystals, the former were

used for native data collection. Data were collected from multiple crystals,

and six sets were selected and merged to form the native data set used for

structure determination. Mercury derivatives were obtained by direct addition

of methyl mercury to the crystallization solution of cysteine mutant crystals.

MALDI-TOF mass spectrometry confirmed 60 to 90% derivatization of crys-

tals prior to data collection. All data were collected at Cornell High Energy

Synchrotron Source (CHESS), station A1, with the Princeton 2K CCD (25).

Data were processed with DENZO and SCALEPACK (26) and the CCP4

package (27). Heavy atom positions were determined with SHELX-97 (28)

and cross-difference Fourier analysis. These positions confirmed the fourfold

noncrystallographic symmetry observed in the self-rotation Patterson func-

tion and allowed the determination of initial orientation matrices. An initial

model (90% complete) was built into a solvent flattened (64% solvent con-

tent), fourfold averaged electron density map with the program O (29). The

tracing of the model was facilitated by the use of the mercury atom positions

as residue markers. L86C was used solely for this purpose. After torsional

refinement (with strict fourfold noncrystallographic symmetry constraints)

with X-PLOR 3.851 (30), this model was used in the anisotropic scaling

[sharpening (31)] of the native data with X-PLOR. The structure factor sigma

values were also rescaled appropriately, and the corrected data were used

for all subsequent procedures. Fourfold averaging, solvent flattening, and

phase extension were applied in DM (32), resulting in a marked improvement

of the electron density that allowed correction of the model and the building

of additional residues. Refinement consisted of rounds of positional (in the

initial stages phase information was also included as a restraint) and grouped

B-factor refinement in X-PLOR. Fourfold noncrystallographic symmetry was

highly restrained with the force constant for positional restraints set as 1000

kcal/mol/Å

. The diffuse ion cloud described in the text was initially modeled

as one or more K

ions and several water molecules; however, the results

were unsatisfactory. Therefore, this and other strong unmodelled density

present in solvent-flattened maps (no averaging included) was Fourier back-

transformed, scaled, and included in the refinement procedure as partial

structure factors. The final model includes amino acids 23 to 119 of each

chain. The following residues were truncated: Arg

to Cb, Ile

to Cg, Arg

to Cb, Glu

to Cb, and Arg

117

to N«. The stereochemistry is strongly

restrained, with no outliers on the Ramachandran plot. The high B-factor

values reflect the intensity decay of the data beyond 4 Å.

Data collection and phasing

Data set

Resolution

(Å)

Redundancy

Completeness

overall/outer

(%)

merge

Phasing

power†

R-Cullis‡

L90C-a 15.0–3.7 3.5 91.3/93.3 0.071 1.61 0.70

L90C-b 15.0–3.7 7.0 91.5/94.1 0.083 1.87 0.50

V93C 15.0–3.7 4.1 98.3/99.1 0.075 1.35 0.63

A32C 15.0–4.0 2.3 84.1/83.8 0.076 1.45 0.66

A29C 15.0–5.0 2.7 73.9/74.0 0.063 1.03 0.85

A42C 15.0–6.5 2.0 90.7/90.3 0.057 0.97 0.81

L86C 30.0–6.0 2.3 58.7/58.9 0.057 — —

Native 30.0–3.2 6.1 93.3% 0.086 15.8 75

Outer Shell 3.3–3.2 2.3 66.6% 0.286 3.9 50

Anisotropic correction

Before sharpening¶

After sharpening¶

Average F.O.Mi

(30.0–3.2 Å)

Average F.O.Mi

(3.4–3.2 Å)

0.76

0.83

0.55

0.64

Refinement Root-mean-square deviation of

Resolution: 10.0–3.2 Å Bond angles: 1.096°

R-cryst.§: 28.0% Bond lengths: 0.005 Å

R-free§: 29.0% Ncs related atoms: 0.006 Å

No. of reflections with F/sF . 2: 12054 B-factor for ncs related atoms: 10 Å

No. of protein atoms: 710 per subunit B-factor for non-bonded atoms: 36 Å

No. of ligand atoms: 1 water, 3 K

ions

Mean B-factor for main-chain atoms: 90 Å

Mean B-factor for side-chain atoms: 110 Å

merge

5SSI2Ij/SI. †Phasing power 5^F

&/^E&. ‡R-Cullis 5SiF

6 F

2F

i/SF

6 F

, only for centric data. §R-cryst. 5SF

p(calc)

/SF

, R-free the

same as R-cryst, but calculated on 10% of data selected in thin resolution shells and excluded from refinement. iFigure of merit. ¶In both cases, fourfold averaging and

solvent flattening were applied; Ij is the observed intensity, I is the average intensity, F

is the root-mean-square heavy-atom structure factor, E is the lack of closure error, F

is the

structure factor for the derivative, F

is the structure factor for the native, F

is the calculated structure factor for the heavy atom, and F

p(calc)

is the calculated native structure factor.

I/sI % of measured

data with I/sI . 2

SCIENCE

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www.sciencemag.org72

ing medium is minimal and therefore the

energy of the cation is highest. Thus, sim-

ple electrostatic considerations allow us to

understand the functional significance of

the cavity and its strategic location. The

cavity overcomes the electrostatic desta-

bilization resulting from the low dielectric

bilayer by simply surrounding an ion with

polarizable water. A second feature of the

channel structure also stabilizes a cat-

ion at the bilayer center. The four pore

helices point directly at the center of the

cavity (Fig. 3, A, B, and D). The amino to

carboxyl orientation of these helices will

impose a negative electrostatic (cation at-

tractive) potential via the helix dipole

effect (18). The ends of the helices are

rather far (;8 Å) from the cavity center,

but all four contribute to the effect.

Therefore, two properties of the structure,

the aqueous cavity and the oriented heli-

ces, help to solve a fundamental physical

problem in biology—how to lower the

electrostatic barrier facing a cation cross-

ing a lipid bilayer. Thus, the diffuse elec-

tron density in the cavity center (Fig. 6C)

likely reflects a hydrated cation cloud

rather than an ion binding site (Fig. 7).

Alternatively, the channel could have

overcome the destabilizing electrostatic

effects of the bilayer center by lining the

entire pore with a polarizable surface, put-

ting ion binding sites along its entire

length. But the structure shows that, with

the exception of the selectivity filter, the

pore lining is mainly hydrophobic, a gen-

eral property of K

channels (Fig. 1). This

conclusion was anticipated by the land-

mark experiments of Armstrong, which

showed that hydrophobic cations bind in

Fig. 3. Views of the tetramer. (A) Stereoview of a

ribbon representation illustrating the three-dimen-

sional fold of the KcsA tetramer viewed from the

extracellular side. The four subunits are distin-

guished by color. (B) Stereoview from another

perspective, perpendicular to that in (A). (C) Rib-

bon representation of the tetramer as an integral-

membrane protein. Aromatic amino acids on the

membrane-facing surface are displayed in black.

(D) Inverted teepee architecture of the tetramer.

These diagrams were prepared with MOLSCRIPT

and RASTER-3D (33).

RESEARCH ARTICLES

www.sciencemag.org

SCIENCE

VOL. 280

3 APRIL 1998 73

The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity

Figures

Citations

The glutamate receptor ion channels

Glutamate Receptor Ion Channels: Structure, Regulation, and Function

Crystal Structure of a Mammalian Voltage-Dependent Shaker Family K + Channel

Dynamic personalities of proteins.

From Ionic Currents to Molecular Mechanisms: The Structure and Function of Voltage-Gated Sodium Channels

References

The CCP4 suite: programs for protein crystallography

MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures

Improved methods for building protein models in electron density maps and the location of errors in these models.

The ionic channels in excitable membranes.

HOLE: a program for the analysis of the pore dimensions of ion channel structural models.

Related Papers (5)

Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution.

X-ray structure of a voltage-dependent K+ channel

Crystal Structure of a Mammalian Voltage-Dependent Shaker Family K + Channel

Ionic channels of excitable membranes

Ion Channels of Excitable Membranes

Frequently Asked Questions (18)

Q1. What contributions have the authors mentioned in the paper "The structure of the potassium channel: molecular basis of k conduction and selectivity" ?

Q2. What was the effect of the averaging on the structure factor?

Q3. What is the effect of the side chains colored green on the TEA binding?

Q4. What is the general property of all selective ion channels?

Q5. What was the only molecule in the structure that was not associated with an ion peak?

Q6. What is the effect of the helices on the electrostatic potential?

Q7. What is the amino acid sequence of the KcsA channel?

Q8. What is the effect of the side chain at the base of the selectivity filter?

Q9. How did the authors determine the ion position of the K1 channel?

Q10. What is the structure of the selectivity filter?

Q11. What is the effect of acidic amino acids on the intracellular and extracellular entryways?

Q12. What is the effect of the second ion on the selectivity filter?

Q13. What is the role of the cerebellum in the acquisition and retention of delay classical conditioning?

Q14. What is the effect of the polarizable surface on the pore?

Q15. What is the appearance of an inverted teepee?

Q16. What was the structure factor used for the refinement procedure?

Q17. Why is the selectivity filter held open?

Q18. Why are K1 channels classified as long pore channels?